Method for selectively transducing pathologic mammalian cells using a tumor suppressor gene

ABSTRACT

A method for transducing a pathologic hyperproliferative mammalian cell is provided by this invention. This method requires contacting the cell with a suitable retroviral vector containing a nucleic acid encoding a gene product having a tumor suppressive function. Also provided by this invention is a method for treating a pathology in a subject caused by the absence of, or the presence of a pathologically mutated tumor suppressor gene.

FIELD OF THE INVENTION

[0001] The present invention generally relates to a method forselectively transducing pathologic hyperproliferative mammalian cells ina heterogeneous cell preparation comprising retroviral-mediatedtransduction of the pathologic cell with a nucleic acid encoding a geneproduct having tumor suppressive function.

[0002] Throughout this application various publications are referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

[0003] The human p53 gene encodes a 53 kilodalton nuclear phosphoprotein(Lane, D. P., et al., Genes and Dev., 4:1-8 (1990); Lee, Y-HP, BreastCancer Res.and Trmt, 19:3-13 (1991); Rotter, V., et al., Adv. Can. Res.,57:257-72 (1991)). The p53 protein was first identified as a cellularprotein in SV40-transformed cells that was tightly bound to the SV40 Tantigen (Lane, D. P., et al. Nature, 278:261-3 (1979)). The wild typep53 gene has the characteristics of a tumor suppressor gene. It issimilar to the prototype of tumor suppressor genes, the retinoblastomagene (RB), in that loss of heterozygosity of the p53 or RB genescharacterizes the phenotype of many types of tumor cells (Hollstein, M.et al., Science, 253:49-51 (1991); Levine, A. J., et al., Biochimica etBiophysica Acta, 1032:119-36 (1990); Levine, A. J., et al., Nature351:453-6 (1991); Weinberg, R. A. Science, 254:1138-46 (1991)). In humanmalignancies associated with p53 alterations, this loss ofheterozygosity usually results from the loss of one allele (allelicdeletion), while the other allele suffers one or more somatic mutations.Unlike RB, however, certain mutations in the p53 gene are capable ofimmortalizing rodent cells and enhancing the tumorigenicity ofestablished cell lines (Jenkins, J. R., et al., Nature, 312:651-4(1984)). Mutant but not wild type p53 can cooperate with the activatedras oncogene in transforming primary rat embryo fibroblasts (Eliyahu,D., et al., Nature, 312(13):646-9 (1984); Parada, L. F., et al., Nature,312:649-51 (1984)). Other events related to tumor progression alsoappear to be associated with the expression of mutant p53. Among theseis differential modulation of the multiple drug resistance gene (MDR1)by wild type as compared to altered p53. In this case, mutant p53specifically stimulates the MDR1 promoter, while wild type p53 exertsrepression (Chin, K-V., et al., Science, 255:459-62 (1992)). Anotherpossible way in which mutant p53 could promote tumorigenesis is byreducing tumor cell responsiveness to transforming growth factor-β, anegative regulator of cell proliferation (Gerwin, B. I., et al., PNASUSA, 89:2759-63 (1992)).

[0004] In addition to the in vitro data described above two animalmodels have been described that implicate p53 in tumor formation.Transgenic mice expressing a mutant p53 gene display a high incidence oflung, bone and lymphoid tumors (Lavigueur, A., et al. Mol. Cell. Biol.,9(9):3982-91 (1989)). In addition, p53-null mice (Donehower, L. A., etal., Nature, 356(19):215-21 (1992)) show an increased risk ofspontaneous neoplasms, the most frequently observed being malignantlymphoma.

[0005] Other data which support the conclusion that mutant p53 plays animportant role in tumorigenesis include re-introduction of the wild typep53 gene into human tumor cell lines which lack p53 expression. In thiscase, wild type p53 can reverse the malignant phenotype as measured bycolony formation in soft agar and tumor formation in nude mice (Chen, P.L., et al., Science, 250:1576-80 (1990); Cheng, J., et al., Can. Res.,52:222-6 (1992); Baker, S. J., et al., Science, 249:912-15 (1990);Isaacs, W. B., et al., Can. Res., 51:4716-20 (1991); Casey, G., et al.,Oncopene, 6(10):1791-7 (1991); Shaw, P., et al., PNAS USA, 89:4495-99(1992); Takahashi, T., et al., Can. Res., 52:2340-3 (1992)). Tumor celltypes which have shown conversion of a non-malignant phenotype as aresult of the introduction of wild type p53 expression include prostate(Isaacs, W. B., et al., supra) , breast (Casey, G. et al. supra), colon(Baker, S J., et al., supra; Shaw, P. et al., supra) lung (Takahashi, T.et al., supra) , and lymphoblastic leukemia (Cheng, J. et al., supra).Other data suggest that introduction of wild type p53 into tumor cellswhich have lost endogenous p53 expression appears to be cytotoxic(Johnson, P. et al., Mol. Cell. Biol., 11(1):1-11 (1991)). In some casesthe re-introduction of wild type p53 may result in programmed celldeath, or apoptosis (Yonish-Rouach, E. et al., Nature, 352:345-7(1991)). The work described above indicates strongly that alteration ofthe wild type p53 gene has a role in multiple aspects of tumorigenesisand that reintroduction of the wild type p53 coding sequence can have anegative regulatory function or cytotoxic effect on malignant cells.

[0006] Clinical data suggest that inactivating mutations in the p53 geneare among the most common types of mutations associated with humanmalignancy (Rotter, V. et al. supra; Nigro, J. M. et al., Nature,342:705-8 (1989); Gaidano, G. et al., PNAS USA, 88:5413-7 (1991); Cheng,J. et al., Mol. Cell. Biol., 10(10):5502-09 (1990)). A classical exampleis the Li-Fraumeni syndrome, a familial syndrome of several neoplasms,including breast cancer, sarcomas and others. Specific mutations in thep53 gene are found in affected members of the family and appear to beassociated with the predisposition to develop early cancers (Malkin, D.et al., Science, 250:1233 (1990); Srivastava, S. et al., Nature, 348:747(1990)). Several laboratories have reported that alterations in the p53gene accompany the evolution of human CML (chronic myelogenous leukemia)to blast crisis (acute phase) (Ahuja, H. et al., J.Clin.Invest.,87:2042-7 (1991); Foti, A. et al., Blood, 77(11):2441-4 (1991);Feinstein, E. et al., PNAS USA, 88:6293-7 (1991)). In one CML patientwho reverted briefly from the acute phase to a second chronic phase, theinactivating point mutation in p53 which appeared concomitantly with theacute phase disappeared and the wild type sequence re-emerged (Foti, A.et al., supra). These data indicate that alterations which inactivatethe tumor suppressive activity of p53 may represent pivotal events inthe progression from the chronic to the acute phase of human CML. Otherclinical data also suggest an important role for p53 mutations indisease progression. These include a number of hematologic neoplasms aswell as solid tumors (Rotter, V. et al. supra; Ahuja, H. et al.,J.Clin.Invest., supra; Ahuja, H. et al., PNAS USA, 86:6783-6787 (1989);Mori, N. et al., Br. J. of Haem., 81:235-240 (1992); Porter, P. L. etal., Am.J.Path., 140(1):145-53 1992)). Recent reports show a dramaticassociation between altered p53 and shortened survival in breast cancer(Thor, A. D. et al., J.Natl. Can. Inst., 84(11):845-55 (1992); Isola, J.et al., J.Natl. Can. Inst.,84(14):1109-14 (1992); Callahan, R. J.Natl.Can.Inst., 84:826-7 (1992)).

SUMMARY OF THE INVENTION

[0007] The present invention generally relates to a method forselectively transducing pathologic hyperproliferative mammalian cellscomprising retroviral-mediated transduction of pathologic cells with anucleic acid encoding a gene product having tumor suppressive function.The methodology provided involves the introduction of a stably expressedtumor suppressor gene into a heterogeneous cell preparation (containingboth normal and pathologic hyperproliferative cells) and, under suitableconditions, selectively transducing phenotypically pathologichyperproliferative cells, suppressing the pathologic phenotype andreinfusing the treated cell preparation into the patient. Also providedby this invention is a method for treating a pathology in a subjectcaused by the absence of, or the presence of a pathologically mutatedtumor suppressor gene.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 shows the tumorigenicity of antibiotics-selected K562 cellsin nude mice. K562 cells were infected with the p53-RV or NCV andselected in hygromycin as described in the legend to Table 3. (A) 5×10⁶K562/p53 or K562/NCV (B) 1×10⁷ K562/p53 or K562/NCV were injectedsubcutaneously into opposite flanks of athymic Balb/c nu/nu mice. Themice were purchased from Simonsen Laboratories, Inc. (Gilroy, Calif.)and maintained in a pathogen-free environment. Once tumors were formed,they were measured weekly until the experiments were terminated.

[0009]FIG. 2 shows delayed tumor formation in nude mice induced by K562cells following a short-term infection with the p53-RV. K562 cells wereinfected with the p53-RV or NCV for 4 hours as described in Example II.The viral supernatant was removed and the cells were injectedimmediately into nude mice as described in Example III.

[0010]FIG. 3 shows delayed tumor formation in nude mice induced by threehuman cell lines following short-term infections with the p53-RV. Thethree human cells lines, H69 (human small-cell lung carcinoma), H128(human small-cell lung carcinoma) and HTB9 (human bladder carcinoma),were obtained from the American Type Culture Collection (ATCC),(Rockville, Md.). The short-term infections using either p53-RV or NCVwere performed as described in Examples II and III.

[0011]FIG. 4 shows lack of toxicity of the p53-RV viral supernatant onnormal murine bone marrow cells at a high multiplicity. Normal mousebone marrow cells were obtained from the femurs of Balb/c mice. Thecells were isolated by ficoll-hypaque gradient, and infected with thep53-RV or NCV at MOI=1(A) or MOI=10(B) for 4 hours. At the end ofinfection, the viral supernatant was removed and the cells were seededin 96-well plates at the density of 5×10⁴ cells per well containingmurine GM-CSF ranging from 0 to 400 units/ml. The cells were incubatedfor 3 days and the number of viable cells were determined by the MTTassay as described by Mossman, T., J. Immunol. Methods, 65:55-63 (1983).

[0012]FIG. 5 graphically depicts the fraction of mice surviving with andwithout treatment with p53RV. Fifty SCID mice were each injected with25×10⁶ K562 cells. Within 50 days post-injection, leukemia developed inthe mice. After day 50, the mice were separated and treated with 2.6×10⁵p53RV and 2.6×10⁵ heat-inactivated p53RV, by the i.p. method.

DETAILED DESCRIPTION OF THE INVENTION

[0013] There are approximately 5,000 bone marrow transplantations (BMT)each year (The BBI Newsletter, 156 (1991)). Most of these are performedon leukemia and lymphoma patients. A growing number of BMT are beingdone to support more intensive therapeutic approaches to breast and lungcancers, as well as for other indications (Droz, J. P. Eur. J. Can.,27:831-35 (1991); Menichella, G. Br.J. Haem., 79:444-50 (1991);Osbourne, C. K. Breast Can. Res. Trtmt., 20:511-14 (1991)).Approximately 30% of these patients are candidates for tumor suppressivegene therapy. This number derives from the observation that about 30% ofcancer patients either do not express the tumor suppressor gene orexpress an inactivated form of the tumor suppressor protein (Hollstein,M. et al., supra) . The preferred embodiments detailed below support theefficaciousness of a retrovirus encoding the human wild type tumorsuppressor gene, p53-RV, in reversing the malignant phenotype of severalleukemia and lymphoma cell lines as measured by abrogation orsubstantial inhibition of colony formation in soft agar assays, and asjudged by reversing/inhibiting the ability of tumor cells to grow innude mice following introduction of the wild type p53 gene.

[0014] For the K562 tumor cell line, which is derived from a chronicmyelogenous leukemia patient in blast crisis (Andersson, L. C. et al.,Int. J. Can., 23:143-7 (1979)) for two human small-cell lung carcinomacell lines (H69 and H128) (Gazdar, A. F. et al,. Can. Res.,40(10):3502-7 (1980)), and for one transitional cell (bladder) carcinomacell line (HTB9) (Takahashi, R. et al., PNAS USA, 88:5257-61 (1991))tumor suppression by p53 can be accomplished with a protocol involvingshort-term infections with the p53-RV. This protocol is completelyconsistent with current clinical methodology used in the preparation ofbone marrow or peripheral blood hematopoietic cells for autologous bonemarrow transplantation (ABMT) (Deisseroth, A. B. et al., Human GeneTherapy, 2:359-376 (1991)).

[0015] The present invention generally relates to an improved method ofgene therapy for “negative purging” of pathologic hyperproliferativecells that contaminate preparations of autologous hematopoietic cellsused for bone marrow reconstitution. As used herein, the term“hyperproliferative cells” includes but is not limited to cells havingthe capacity for autonomous growth, i.e., existing and reproducingindependently of normal regulatory mechanisms. Hyperproliferativediseases may be categorized as pathologic, i.e., deviating from normalcells, characterizing or constituting disease, or may be categorized asnon-pathologic, i.e., deviation from normal but not associated with adisease state. Pathologic hyperproliferative cells are characteristic ofthe following disease states, thyroid hyperplasia—Grave's Disease,psoriasis, benign prostatic hypertrophy, Li-Fraumeni syndrome includingbreast cancer, sarcomas and other neoplasms, bladder cancer, coloncancer, lung cancer, various leukemias and lymphomas. Examples ofnon-pathologic hyperproliferative cells are found, for instance, inmammary ductal epithelial cells during development of lactation and alsoin cells associated with wound repair. Pathologic hyperproliferativecells characteristically exhibit loss of contact inhibition and adecline in their ability to selectively adhere which implies a change inthe surface properties of the cell and a further breakdown inintercellular communication. These changes include stimulation to divideand the ability to secrete proteolytic enzymes. The present inventionwill allow for high dose chemotherapy and/or radiation therapy, followedby autologous bone marrow reconstitution with hematopoietic cellpreparations in which phenotypically pathologic cells have beenreconstituted with a normal tumor suppressor gene. Application of thepresent invention will result in diminished patient relapses which occuras a result of reinfusion of pathologic hyperproliferative cellscontaminating autologous hematopoietic cell preparations.

[0016] More specifically, the present invention relates to a method fordepleting a suitable sample of pathologic mammalian hyperproliferativecells contaminating hematopoietic precursors during bone marrowreconstitution via the introduction of a stably-expressed wild typetumor suppressor gene into the cell preparation (whether derived fromautologous peripheral blood or bone marrow). As used herein, a “suitablesample” is defined as a heterogeneous cell preparation obtained from apatient, e.g., a mixed population of cells containing bothphenotypically normal and pathogenic cells. An example of a wild typetumor suppressor gene is the p53 gene, the coding sequence has beendescribed by Chen et al. supra and is shown in Table 1. TABLE 1 V*SHRPGSR* LLGSG DTLRS GWBRA FHDGD TLPWI GSQTA          50 FRVTA MEEPQ SDPSVEPPLS QETFS DLWKL LPENN VLSPL PSQAM DDLML         100 SPDDI EQWFT EDPGPDEAPR MPEAA PPVAP APAAP TPAAP APAPS WPLSS         150 SVPSQ KTYQG SYGFRLGFLH SGTAK SVTCT YSPAL NKMFC QLAKT CPVQL         200 WVDST PPPGT RVRAMAIYKQ SQHMT EVVRR CPHHE RCSDS DGLAP PQHLI         250 RVEGN LRVEY LDDRNTFRHS VVVPY EPPEV GSDCT TIHYN YMCNS SCMGG         300 MNRRP ILTII TLEDSSGNLL GRNSF EVRVC ACPGR DRRTE EENLR KKGEP         350 HHELP PGSTK RALPNNTSSS PQPKK KPLDG EYFTL QIRGR ERFEM FRELN         400 EALEL KDAQA GKEPGGSRAH SSHLK SKKGQ STSRH KKLMF KTEGP DSD*

[0017] The preferred delivery system for the wild type tumor suppressorgene is a replication-incompetent retroviral vector. As used herein, theterm “retroviral” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thecoding sequence into dividing cells. As used herein, the terms“replication-incompetent” is defined as the inability to produce viralproteins, precluding spread of the vector in the infected host cell. Anexample of such vector is p53-RV, which has been described in detail byChen et al. supra and is shown in Table 2. TABLE 2 General Schematic ofp53-Retrovirus (p53-RV)

[0018] Another example of a replication-incompetent retroviral vector isLNL6 (Miller, A. D. et al., BioTechnigues 7:980-990 (1989)). Themethodology of using replication-incompetent retroviruses forretroviral-mediated gene transfer of gene markers is well established(Correll, P. H. et al., PNAS USA, 86:8912 (1989); Bordignon, C. et al.,PNAS USA, 86:8912-52 (1989); Culver, K. et al., PNAS USA, 88:3155(1991); Rill, D. R. et al.,Blood, 79(10):2694-700 (1992)). Clinicalinvestigations have shown that there are few or no adverse effectsassociated with the viral vectors (43: Anderson, Science, 256:808-13(1992)). However, these methods have been limited to transfers of “genemarkers” such as the neomycin gene that merely function as “trackingagents” for marking malignant cells before, and locating malignant cellsafter, reinfusion of bone marrow, however, the transduction of genemarkers confers little clinical benefit to the affected patient who doesnot receive protection against subsequent relapse (Rill, D. R. et al.Blood, sudra). The subject invention eliminates the necessity of thetime consuming procedure of transducing cell samples with a selectablemarker gene, such as neomycin, to identify pathologic cells tofacilitate subsequent attempts to remove those cells before reinfusioninto the patient.

[0019] Other vectors are suitable for use in this invention and will beselected for effecient delivery of the nucleic acid encoding the tumorsuppressor gene. The nucleic acid can be DNA, cDNA or RNA.

[0020] The subject invention provides a “shotgun” procedure whereby thecell sample is contacted with a retroviral vector in the absence ofselective medium that does not necessarily contain a selectable markergene, but notwithstanding, possesses the ability to simultaneouslyselectively target and transduce only the pathologic cell population inthe heterogeneous cell preparation. Other methods of efficient deliveryor insertion of a gene of interest into a cell are well known to thoseof skill in the art and comprise various molecular cloning techniques.As used herein, the terms “insertion or delivery” encompass methods ofintroducing an exogenous nucleic acid molecule into a cell.

[0021] A variety of techniques have been employed in an attempt todeplete marrow of pathologic hyperproliferative cells before reinfusion,utilizing “purging” methods, e.g., monoclonal antibodies or chemotoxins(Kaizer H. et al., Blood, 65:1504 (1985); Gorin, N. C. et al., Blood,67:1367 (1986); De Fabritiis, P. et al., Bone Marrow Transplant, 4:669(1989)). As used herein, the term “pathologic” includes abnormalitiesand malignancies induced by mutations and failures in the geneticregulatory mechanisms that govern normal differentiation that are notthe result of gene loss or mutation. These techniques, however, have notresulted in reduced relapse rates, and have consistently resulted indamaging normal marrow progenitor cells (Kaizer H. et al., supra; Gorin,N. C. et al., supra; De Fabritiis, P. et al., supra). The presentinvention addresses the aforementioned inadequacies and confers relatedadvantages as well. These advantages include: (a) the use of arecombinant retroviral vector that does not require a selectable markergene in combination with a short-term infection in the absence ofselective medium eliminating the time consuming procedure traditionallyemployed to “selectively mark” the target cells before any “purging” ofsuch cells is attempted, thereby dramatically reducing the timetraditionally required for preparing hematopoietic cells fortransplants; and (b) the retroviral mediated delivery methodology of thesubject invention offers selective targeting of pathologichyperproliferative cells in resting cultures of hematopoietic cells as aresult of the higher infection frequency by the retroviral deliverysystem into actively dividing tumor cells (Miller et al., Mol. Cell.Biol., 10(8):4239-42 (1990)).

[0022] The ex-vivo introduction of a wild type tumor suppressor gene,via an efficient delivery system into pathologic hyperproliferativecells contaminating peripheral blood- or marrow-derived autologoushematopoietic cells will facilitate suppression of thehyperproliferative phenotype, by inducing transformation of the cell toa mature or benign phenotype or, alternatively, by inducing apoptosis orprogrammed cell death, thereby allowing patients receiving ABMT to havea longer, relapse-free survival. As used herein, the term “mature orbenign cell” refers to the phenotypic characteristic of inability toinvade locally or metastasize.

[0023] This invention further provides a method for transducing apathologic hyperproliferative mammalian cell by contacting the cell witha suitable retroviral vector containing a nucleic acid encoding a geneproduct having a tumor suppressive function, under suitable conditionssuch that the cell is transduced. In one embodiment, the gene product isexpressed by a tumor suppressor gene and the tumor suppressor gene canbe, but is not limited to wild type p53 gene, retinoblastoma gene RB,Wilm's tumor gene WT1 or colon carcinoma gene DCC. Additionally, thenucleic acid is DNA, cDNA or RNA.

[0024] The suitable conditions for contacting can be by infecting thesample cells in the absence of selective medium. “Suitable retroviralvector” has been defined above. This method is particularly useful whenthe pathological cells being contacted are prostate cells, psoriaticcells, thyroid cells, breast cells, colon cells, lung cells, sarcomacells, leukemic cells or lymphoma cells.

[0025] The suitable time period for contacting can be less than aboutten hours, or more specifically, about four hours. Transduction can beknown to be complete, for example, when the hyperproliferative phenotypeis characterized by the transduced cell expressing a mature or benignphenotype or by apoptosis or death of the transduced cell. This methodhas been shown to reduce tumor formation or tumorigenicity in a subject.

[0026] This method can be practiced ex vivo or in vivo. The practice ofthe ex vivo method is described above. When the method is practiced invivo, the retroviral vector can be added to a pharmaceuticallyacceptable carrier and systemically administered to the subject. In oneembodiment, the subject is a mammal, such as a human patient. Acceptable“pharmaceutical carriers” are well known to those of skill in the artand can include, but not be limited to any of the standardpharmaceutical carriers, such as phosphate buffered saline, water andemulsions, such as oil/water emulsions and various types of wettingagents.

[0027] As used herein, the term “administering” for in vivo purposesmeans providing the subject with an effective amount of the vector,effective to inhibit hyperproliferation of the target cell. Methods ofadministering pharmaceutical compositions are well known to those ofskill in the art and include, but are not limited to, microinjection,intravenous or parenteral administration. Administration can be effectedcontinuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and will varywith the vector used for therapy, the purpose of the therapy, the cellor tumor being treated, and the subject being treated.

[0028] The following examples are intended to illustrate, not limit thisinvention.

EXAMPLE I Introduction of the D53-RV into Leukemia or Lymphoma-DerivedCell Lines Suppresses the Malignant Phenotype as Measured by ColonyFormation in Soft Agar

[0029] The retroviral vector carrying the human wild type p53-cDNA hasbeen described (Chen et al. supra). p53-RV, an amphotropic retrovirus,is capable of infecting a wild range of human cell types (see below).This feature provides an advantage for ex vivo therapy of humanleukemias, because the viral vector can deliver the wild type p53-cDNAinto a number of different leukemic or other cell types, including tumorcells from solid tumors which may metastasize to marrow. The results ofsoft-agar assays using three leukemia or lymphoma cell lines followingviral infection and antibiotic selection are shown in Table 3. In allthree cases (HL-60, acute promyelocytic leukemia, p53-negative; Hut 78,acute T cell lymphoma, p53-negative; and Molt 3, acute lymphoblasticleukemia, mutant p53-positive) the introduction of wild type p53 by thep53-RV resulted in either a reduction or elimination of colony formationin soft agar. TABLE 3 Cell Line No. of Cells Seeded Plating EfficiencyHL-60 5 × 10⁵ TMTC HL-60/T* 5 × 10⁵  4.7% HL-60    10⁵   43% HL-60/T*   10⁵   0% HL-60 5 × 10⁴   55% HL-60/T* 5 × 10⁴   0% Hut 78    10⁵ 9.4% Hut 78/I#    10⁵ 0.39% Hut 78 5 × 10⁴  9.2% Hut 78/I# 5 × 10⁴   0%Molt 3    10⁵ 11.7% Molt 3/I#    10⁵  1.5%

[0030] The human leukemic cell lines, HL-60, Hut 78 and Molt 3, wereobtained from American Type Culture Collection (ATCC). The cell linesHut 78 and Molt 3 were infected with the p53-RV and the HL-60 cell linewas transfected with p53-RV DNA. The p53-RV containing the wild type p53cDNA isolated from human fetal brain and Moloney murine leukemia viralvector has been described by Chen et al., supra. This virus also carriesthe hygromycin resistant gene whose expression is driven by the Roussarcoma virus (RSV) promoter sequence. The murine NIH3T3-derivedpackaging cell line, PA12 (Chen et al., supra), produces the p53-RV withtiters ranging from 1×10⁵ to 1×10⁶ virus per ml.

[0031] Viral infections were carried out overnight in he presence of 4μg/ml polybrene. At the end of infections, 4 ml of fresh media wereadded to 2 ml of each infection mixture. The cells were selected in thepresence of 400 μg/ml hygromycin 2 days after infection.

[0032] Infected cells grew to confluency in 2-3 weeks followinghygromycin selection. For the soft-agar assay, the cells were seeded in6-well plates at the cell densities ranging from 103 to 105 in 0.33%agar as described (Chen et al., supra). Colonies consisting of more than50 cells were scored 2 weeks later.

EXAMPLE II Suppression of Colony Formation in Soft Agar by K562 (HumanChronic Myelogenous Leukemia) Cells Following a Short-Term InfectionWith p53-RV

[0033] Mammalian cells infected with a retroviral vector carrying anantibiotic marker are usually pre-selected in vitro before testing fortumorigenicity in soft agar or in nude mice (Chen et al., supra, Chenget al., supra). Because this process takes about three weeks, it wouldbe cumbersome and expensive to pursue in the clinic. To mimic moreclosely the clinical situation in which tumor suppressor gene therapymay be applied during bone marrow purging, K562 cells were infected withp53-RV for four hours in vitro, then immediately tested their ability toform colonies in soft agar without any selection. A retroviral vectoridentical to p53-RV, but with the p53 coding sequence deleted, was usedas a control (see Table 4). This vector is designated NCV (NegativeControl Virus). As shown in Table 4, p53-RV decreased colony formationby infected K562 cells in a dose-dependent manner. At a multiplicity ofinfection, (MOI) of 1, the plating efficiencies of the p53 andNCV-infected cells were similar. However, at MOI of 3 and 10, there wasa marked decrease in the plating efficiency of the p53-RV infectedcells. The plating efficiencies of the NCV-infected K562 cells weresimilar at all three multiplicities of infection. The latter resultsuggests that the does-dependent reduction in tumor cell colonyformation observed with increasing doses of p53-RV was due tointroduction of the wild type p53. Furthermore, the result with NCVindicates that there is little non-specific toxicity associated with theretroviral infection up to MOI of 10, as measured by this assay. TABLE 4Virus No. of Cells Plating Efficiency Infection Seeded M.O.I. (ColonyNo.) p53-RV    10⁴ 1 3.10% (310) 3 0.52% (52) 10   0% (0) Control RV   10⁴ 1 4.30% (430) 3 5.30% (530) 10 3.40% (340) p53-RV 5 × 10³ 1 4.40%(220) 3 1.80% (90) 10 0.25% (13) Control RV 5 × 10³ 1 4.70% (235) 34.10% (205) 10 6.50% (325)

[0034] Human chronic myelogenous leukemia (CML)-derived cell line, K562,was obtained from ATCC, Accession No. CCL243. To perform the short-terminfections, K562 cells were infected with the p53-RV or NCV for 4 hoursas described in Example I. Multiplicity of infection (MOI) wasdetermined from the titer of the viral stocks and K562 cell number. Atthe end of infection, the viral supernatant was removed by pelleting thecells, and the concentrated cells were used immediately in the soft-agarassay as described in Example I.

[0035] To construct NCV, the plasmid containing the p53 viral genome waspartially digested with BamHI, ligated, and used to transform E. coli.The plasmid with the deleted p53 gene was then selected by restrictionenzyme analysis of mini-lysate DNA. This DNA was used totransfect/infect PsiCRIP packaging cell line as described (Danos, O. etal., PNAS USA, 85:6460-64 (1988)). The viral stock, termed negativecontrol virus (NCV), was produced in PsiCRIP packaging cell line (56:Danos et al., supra) with a titer of about 2×10⁵ virus per ml.

EXAMPLE III Tumorigenicity of K562 Chronic Myelogenous Leukemia CellsFollowing Infection by P53-RV and Selection for Hyaromycin-ResistantCells

[0036] To further broaden the efficacy experiments in relevant humantumor cell lines, K562 leukemic cells were infected with the p53-RV andhygromycin-resistant colonies (K562/p53) were tested for tumorigenicityin nude mice. When 5×10⁶ K562/p53 cells were injected subcutaneouslyinto nude mice, no tumor formation was observed. In contrast, acomparable number of K562/NCV cells produced tumors in all five animalstested (FIG. 1A). When 1×10⁷ tumor cells were injected, the p53-RVinfected cells produced visible tumors, although much smaller than thoseinduced by the NCV-infected cells (FIG. 1B). It is likely that thelesions which formed on the flank of the animal injected with K562/p53were induced by those cells which had lost expression of the wild typep53 gene (Johnson et al., supra). This conclusion is supported by theinability to detect p53 protein or transcripts in hygromycin selectedclones after only a few passages in vitro (data not shown).

EXAMPLE IV Tumorigenicity in Nude Mice of K562 Cells Following aShort-Term Infection with p53-RV

[0037] To further assess the tumor suppressive activity of the wild typep53 gene in K562 cells, and to determine whether a short-term infectionprotocol would be feasible for potential therapy of leukemias andlymphomas, K562 CML cells were co-incubated with p53-RV for four hoursbefore testing for the malignant phenotype as determined by subcutaneoustumor formation in nude mice. Following a short-term infection by thep53-RV or the NCV, K562 cells were injected bilaterally into nude mice.In three separate experiments, substantial suppression of tumorformation on the flank injected with K562 exposed to the p53-RV wasobserved (FIG. 2).

EXAMPLE V Growth Suppressive Activity of P53-RV on Other Human TumorCell Types

[0038] While the major target for clinical trials consists of leukemiaand lymphoma patients, other cancer patients are currently underconsideration for clinical trials involving marrow reconstitution(Miller, C. W. et al., Can. Res., 52:1695-8 (1992); Takahashi, T. etal., OncoQene, 6:1775-8 (1991); Takahashi, T. et al., Science,491-4(1989)). FIG. 3 demonstrates that short-term infections of twosmall-cell lung carcinoma cell lines (H69 in FIG. 3A; H128 in FIG. 3B)lead to substantial inhibition of tumor growth in nude mice. Inaddition, a similar experiment was performed with a human transitionalcell (bladder) carcinoma cell line (HTB-9 in FIG. 3C). In contrast,tumor cells infected with NCV grow rapidly in this tumor model (FIGS.3A-C).

EXAMPLE VI Preliminary in Vitro Toxicity Studies

[0039] A critical issue for clinical application of the p53-RV has to dowith whether introduction of the p53 coding sequence under control of amurine retroviral LTR may inhibit proliferation of normal bone marrowcells. Preliminary studies suggest that such inhibition is not an issuein this system. To determine toxicity of the p53-RV, it was investigatedwhether exposure of normal bone marrow cells to p53-RV under conditionssimilar to those employed for a short-term infection of K562 leukemiccells would have an effect on the response of normal bone marrow cellsto GM-CSF. A three-day proliferation assay and a methylcellulose colonyforming assay using either murine or human normal bone marrow cells wereemployed to ascertain such response. FIG. 5 shows that exposure ofmurine bone marrow cells at either a MOI of 1.0 or 10.0 has no effect ontheir proliferation in response to GM-CSF. In addition, when eitherhuman (Table 3) or murine (data not shown) bone marrow cells were testedin a GM-CSF dependent colony forming assay, no effect on normal marrowprogenitor colony forming units following exposure to the p53-RV ascompared to NCV or mock infected controls was observed.

[0040] Normal human bone marrow cells were isolated by ficoll-hypaquegradient. These cells were incubated with the p53-RV, NCV, or growthmedia in the presence of 4 μg/ml polybrene for 4 hours. At the end ofincubation, the cells were pelleted, and 1×10⁶ cells per well wereplated in 6-well plates containing 0.8% methylcellulose. Colonies largerthan 50 cells per colony were scored 14 days later. TABLE 5 COLONYNUMBER rHuGMCSF Infection None 0.02 ng/ml 0.04 ng/ml Control 1 18 21p53-RV(0.1) 0 16 28 NCV(0.1) 0 11 18 MOCK(0.1) 2 12 17 p53-RV(1.0) 2 1523 NCV(1.0) 4 9 18 MOCK(1.00) 0 25 18

EXAMPLE VII Negative Purging of Small Cell Lung Cancer Cells (H69) Froma Preparation of Human Bone Marrow

[0041] Increasing quantities of small-cell lung cancer cell line H69were added to human bone marrow cells. These cells were subjected to 3two hour cycles of infection with p53-RV at a M.O.I. of 3. Afterinfection the cells were pelleted and plated in methylcellulose. Colonyformation is shown in Table 6. Suppression of tumor cell colonyformation is evidenced in the p53-RV treated cultures, but is absent inthe mock infected cultures. There is no evidence of suppression of bonemarrow colony formation units in either case. TABLE 6 Negative Purgingof Small Cell Lung Cancer Cells (H69) From a Preparation of Human BoneMarrow 0 0.1 1.0 10.0 H69/HBMC% A. Mock Colonies H69 0 53 100 475 HMBC +Growth 199 219 235 266 Factors B. P53-RV Colonies H69 0 1 11 181 HMBC +Growth 165 223 182 273 Factors HBMC + Growth 81 48 84 171

EXAMPLE VIII Mixing Experiment to Study “Bystander Effect”

[0042] Five (5)×10⁷ K562 cells (obtained from the American Type CultureCollection (ATCC)) were infected overnight with p53RV at a MOI equals 1in RPMI medium containing 4 ug/ml polybrene (Sigma). Twenty-five nudemice were divided into 5 groups, with 5 animals per group. Every mousein group 1 was infected subcutaneously with 5×10⁶ of p53RV infectedcells. The ratio of infected to uninfected cells=1:0. Every mouse ingroup 2 was injected subcutaneously with a mixture of 2.5×10⁶ infectedcells and 2.5×10⁶ uninfected cells. The ratio of infected to infectedcells=1:1. Every mouse in group 3 was injected subcutaneously with amixture of 1.5×10⁶ infected cells and 3×10⁶ uninfected cells. The ratioof infected to uninfected cells=1:2. Every mouse in group 4 was injectedsubcutaneously with a mixture of 0.45×10⁶ infected and 4.5×10⁶uninfected cells (infected:uninfected=1:10). All the mice in group 5were injected subcutaneously with 5×10⁶ uninfected cells(infected:uninfected=0:1). Nude mice were observed for tumor growth andsurvival time. Results of the study are summarized below. Ratio TumorSurvival Group infected:uninfected Formation Status^(b) 1 1:0 — Aliveand healthy at 200 days 2 1:1 — Alive and healthy at 200 days 3 1:2 —Alive and healthy at 200 days 4 1:10 — Alive and healthy at 200 days 50:1 +^(a) All died within 90 days

[0043] This experiment shows that treatment with p53RV, even at a MOI ofless than 1, inhibits tumor formation or “tumorigenicity” in nude mice.

EXAMPLE IX Effect of Intraperitoneal Injection of p53RV in K562 BearingSCID Mice

[0044] Fifty (50) SCID mice were each injected i.p. with 25×10⁶ K562cells (ATCC). Within 50 days post-injection, leukemia developed in themice. The mice were then randomly separated into 3 groups and treated asoutlined below:

[0045] Group 1: injected i.p. with RPMI media on day 50

[0046] Group 2: injected i.p. with 1 ml heat-inactivated p53RV (originaltiter=2.6×10⁵ virus/ml, titer below detection limit after inactivation)on day 50

[0047] Group 3: injected i.p. with 1 ml p53RV (2.6×10⁵ virus/ml) on day50

[0048]FIG. 5 shows that mice treated with p53RV survived over twice aslong as mice treated with heat-inactivated virus or control mice. Thus,systemic treatment with the retroviral vector containing the tumorsuppressor gene p53 reduced tumorigenicity in the mice and prolongedsurvival time.

[0049] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

We claim:
 1. A method for transducing a pathologic hyperproliferativemammalian cell comprising contacting the cell with a suitable retroviralvector containing a nucleic acid encoding a gene product having a tumorsuppressive function, under suitable conditions such that the cell istransduced.
 2. The method of claim 1, wherein the gene product isexpressed by a tumor suppressor gene.
 3. The method of claim 2, whereinthe tumor suppressor gene is wild type p53 gene, retinoblastoma gene RB,Wilm's tumor gene WT1 or colon carcinoma gene DCC.
 4. The method ofclaim 1, wherein the suitable conditions are infecting the sample cellsin the absence of selective medium.
 5. The method of claim 1, whereinthe suitable retroviral vector lacks a selectable marker gene.
 6. Themethod of claim 1, wherein the suitable retroviral vector isreplication-incompetent.
 7. The method of claim 1, wherein thepathological cells are prostate cells, psoriatic cells, thyroid cells,breast cells, colon cells, lung cells, sarcoma cells, leukemic cells orlymphoma cells.
 8. The method of claim 1, wherein the suitable timeperiod is less than about ten hours.
 9. The method of claim 8, whereinthe time period is about four hours.
 10. The method of claim 1, whereinsuppressing the hyperproliferative phenotype is characterized by thetransduced cell expressing a mature or benign phenotype.
 11. The methodof claim 1, wherein suppressing the hyperproliferative phenotype ischaracterized by apoptosis or death of the transduced cell.
 12. Themethod of claim 1, wherein the contacting is effected ex vivo.
 13. Themethod of claim 1, wherein the contacting is effected in vivo.
 14. Themethod of claim 1, wherein the nucleic acid is RNA.
 15. The method ofclaim 1, wherein the mammal is a human.
 16. A method for treating apathology in a subject caused by the absence of a tumor suppressor geneor the presence of a pathologically mutated tumor suppressor genecomprising administering to the subject an effective amount of asuitable retroviral vector containing a nucleic acid encoding a geneproduct having a tumor suppressive function, under suitable conditions.17. The method of claim 16, wherein the gene product is expressed by atumor suppressor gene.
 18. The method of claim 17, wherein the tumorsuppressor gene is wild type p53 gene, retinoblastoma gene RB, Wilm'stumor gene WT1 or colon carcinoma gene DCC.
 19. The method of claim 16,wherein the suitable retroviral vector is replication-incompetent. 20.The method of claim 16, wherein the absence or presence of apathologically mutated tumor suppressor gene causes a cell tohyperproliferate.
 21. The method of claim 20, wherein thehyperproliferative cell is a prostate cell, a psoriatic cell, a thyroidcell, a breast cell, a colon cell, a lung cell, a sarcoma cell, aleukemic cell or a lymphoma cell.
 22. The method of claim 21, whereinthe treating of the hyperproliferative cell is characterized byapoptosis or death of the cell.
 23. The method of claim 16, wherein thecontacting is effected in vivo.
 24. The method of claim 16, wherein thenucleic acid is RNA.